Microtubule regrowth assay

C2C12 cells were treated with 5 µM nocodazole (Sigma-Aldrich, Cat# M1404) in culture media for 3 hr at 37°C to depolymerize microtubules. To observe microtubule regrowth, nocodazole-containing medium was removed, cells were rinsed three times with cold medium, and either fixed (0 min time point) with 4% formaldehyde in PBS for 10 min or immediately transferred to 37°C pre-warmed culture media for the desired length of time followed by formaldehyde fixation. Myotubes were extracted with 1% Triton X-100 in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, pH 6.9) for 30 s at room temperature prior to fixation.